CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

ScSmOP: a universal computational pipeline for single-cell single-molecule multiomics data analysis.

Single-cell multiomics techniques have been widely applied to detect the key signature of cells. These methods have achieved a single-molecule resolution and can even reveal spatial localization. These emerging methods provide insights elucidating the features of genomic, epigenomic and transcriptomic heterogeneity in individual cells. However, they have given rise to new computational challenges in data processing. Here, we describe Single-cell Single-molecule multiple Omics Pipeline (ScSmOP), a universal pipeline for barcode-indexed single-cell single-molecule multiomics data analysis. Essentially, the C language is utilized in ScSmOP to set up spaced-seed hash table-based algorithms for barcode identification according to ligation-based barcoding data and synthesis-based barcoding data, followed by data mapping and deconvolution. We demonstrate high reproducibility of data processing between ScSmOP and published pipelines in comprehensive analyses of single-cell omics data (scRNA-seq, scATAC-seq, scARC-seq), single-molecule chromatin interaction data (ChIA-Drop, SPRITE, RD-SPRITE), single-cell single-molecule chromatin interaction data (scSPRITE) and spatial transcriptomic data from various cell types and species. Additionally, ScSmOP shows more rapid performance and is a versatile, efficient, easy-to-use and robust pipeline for single-cell single-molecule multiomics data analysis.

Multiplex chromatin interactions with single-molecule precision.

The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus1. Advanced 3D genome-mapping methods that combine proximity ligation and high-throughput sequencing (such as chromosome conformation capture, Hi-C)2, and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET)3, have revealed topologically associating domains4 with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription5-7. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C8 potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis9 but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.

MCIBox: a toolkit for single-molecule multi-way chromatin interaction visualization and micro-domains identification.

The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies not only offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for multi-way chromatin interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell-cycle-associated genes. Taken together, MCIBox represents an invaluable tool for the study of multiple chromatin interactions and inaugurates a previously unappreciated view of 3D genome structure.

Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities.

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.

Inhibition of Laser-Induced Choroidal Neovascularization by Hematoporphyrin Dimethylether-Mediated Photodynamic Therapy in Rats.

This study aimed to investigate the effect of hematoporphyrin dimethylether (HDME)-mediated photodynamic therapy for laser-induced choroidal neovascularization (CNV) in adult Brown Norway rats. HDME was administered via tail vein at 14 d after the laser photocoagulation, and the rats received irradiance with a laser light at 570 nm at 15 min after injection. CNV was evaluated by fundus photography, fundus fluorescein angiography, optical coherence tomography, and hematoxylin and eosin staining. We found that CNV was occurred at 7 d after photocoagulation and reaching peak activity at 14 d after photocoagulation. There is a significant reduction in the total area of the fluorescein leakage and the number of strong fluorescein leakage spots on 7 d after HDME-mediated photodynamic therapy (PDT). The results suggest that HDME-mediated PDT inhibits laser-induced CNV in rats, representing a promising therapy for wet age-related macular degeneration.

Photoinitiated polymerization of cationic acrylamide in aqueous solution: synthesis, characterization, and sludge dewatering performance.

A copolymer of acrylamide (AM) with acryloyloxyethyl trimethyl ammonium chloride (DAC) as the cationic monomer was synthesized under the irradiation of high-pressure mercury lamp with 2,2-azobis(2-amidinopropane) dihydrochloride (V-50) as the photoinitiator. The compositions of the photoinduced copolymer were characterized by Fourier transform infrared spectra (FTIR), ultraviolet spectra (UV), and scanning electron microscope (SEM). The effects of 6 important factors, that is, photo-initiators concentration, monomers concentration, CO(NH2)2 (urea) concentrations, pH value, mass ratio of AM to DAC, and irradiation time on the molecular weight and dissolving time, were investigated. The optimal reaction conditions were that the photo-initiators concentration was 0.3%, monomers concentration was 30 wt.%, irradiation time was 60 min, urea concentration was 0.4%, pH value was 5.0, and mass ratio of AM to DAC was 6 : 4. Its flocculation properties were evaluated with activated sludge using jar test. The zeta potential of supernatant at different cationic monomer contents was simultaneously measured. The results demonstrated the superiority of the copolymer over the commercial polyacrylamide as a flocculant.

Specific multivalent molecules boost CRISPR-mediated transcriptional activation.

CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.

3D chromatin structures associated with ncRNA roX2 for hyperactivation and coactivation across the entire X chromosome.

The three-dimensional (3D) organization of chromatin within the nucleus is crucial for gene regulation. However, the 3D architectural features that coordinate the activation of an entire chromosome remain largely unknown. We introduce an omics method, RNA-associated chromatin DNA-DNA interactions, that integrates RNA polymerase II (RNAPII)-mediated regulome with stochastic optical reconstruction microscopy to investigate the landscape of noncoding RNA roX2-associated chromatin topology for gene equalization to achieve dosage compensation. Our findings reveal that roX2 anchors to the target gene transcription end sites (TESs) and spreads in a distinctive boot-shaped configuration, promoting a more open chromatin state for hyperactivation. Furthermore, roX2 arches TES to transcription start sites to enhance transcriptional loops, potentially facilitating RNAPII convoying and connecting proximal promoter-promoter transcriptional hubs for synergistic gene regulation. These TESs cluster as roX2 compartments, surrounded by inactive domains for coactivation of multiple genes within the roX2 territory. In addition, roX2 structures gradually form and scaffold for stepwise coactivation in dosage compensation.

MCI-frcnn: A deep learning method for topological micro-domain boundary detection.

Chromatin structural domains, or topologically associated domains (TADs), are a general organizing principle in chromatin biology. RNA polymerase II (RNAPII) mediates multiple chromatin interactive loops, tethering together as RNAPII-associated chromatin interaction domains (RAIDs) to offer a framework for gene regulation. RAID and TAD alterations have been found to be associated with diseases. They can be further dissected as micro-domains (micro-TADs and micro-RAIDs) by clustering single-molecule chromatin-interactive complexes from next-generation three-dimensional (3D) genome techniques, such as ChIA-Drop. Currently, there are few tools available for micro-domain boundary identification. In this work, we developed the MCI-frcnn deep learning method to train a Faster Region-based Convolutional Neural Network (Faster R-CNN) for micro-domain boundary detection. At the training phase in MCI-frcnn, 50 images of RAIDs from Drosophila RNAPII ChIA-Drop data, containing 261 micro-RAIDs with ground truth boundaries, were trained for 7 days. Using this well-trained MCI-frcnn, we detected micro-RAID boundaries for the input new images, with a fast speed (5.26 fps), high recognition accuracy (AUROC = 0.85, mAP = 0.69), and high boundary region quantification (genomic IoU = 76%). We further applied MCI-frcnn to detect human micro-TADs boundaries using human GM12878 SPRITE data and obtained a high region quantification score (mean gIoU = 85%). In all, the MCI-frcnn deep learning method which we developed in this work is a general tool for micro-domain boundary detection.

A Low-Producing Haplotype of Interleukin-6 Disrupting CTCF Binding Is Protective against Severe COVID-19.

Interleukin6 (IL-6) is a key driver of hyperinflammation in COVID-19, and its level strongly correlates with disease progression. To investigate whether variability in COVID-19 severity partially results from differential IL-6 expression, functional single-nucleotide polymorphisms (SNPs) of IL-6 were determined in Chinese COVID-19 patients with mild or severe illness. An Asian-common IL-6 haplotype defined by promoter SNP rs1800796 and intronic SNPs rs1524107 and rs2066992 correlated with COVID-19 severity. Homozygote carriers of C-T-T variant haplotype were at lower risk of developing severe symptoms (odds ratio, 0.256; 95% confidence interval, 0.088 to 0.739; P = 0.007). This protective haplotype was associated with lower levels of IL-6 and its antisense long noncoding RNA IL-6-AS1 by cis-expression quantitative trait loci analysis. The differences in expression resulted from the disturbance of stimulus-dependent bidirectional transcription of the IL-6/IL-6-AS1 locus by the polymorphisms. The protective rs2066992-T allele disrupted a conserved CTCF-binding locus at the enhancer elements of IL-6-AS1, which transcribed antisense to IL-6 and induces IL-6 expression in inflammatory responses. As a result, carriers of the protective allele had significantly reduced IL-6-AS1 expression and attenuated IL-6 induction in response to acute inflammatory stimuli and viral infection. Intriguingly, this low-producing variant that is endemic to present-day Asia was found in early humans who had inhabited mainland Asia since ∼40,000 years ago but not in other ancient humans, such as Neanderthals and Denisovans. The present study suggests that an individual's IL-6 genotype underlies COVID-19 outcome and may be used to guide IL-6 blockade therapy in Asian patients. IMPORTANCE Overproduction of cytokine interleukin-6 (IL-6) is a hallmark of severe COVID-19 and is believed to play a critical role in exacerbating the excessive inflammatory response. Polymorphisms in IL-6 account for the variability of IL-6 expression and disparities in infectious diseases, but its contribution to the clinical presentation of COVID-19 has not been reported. Here, we investigated IL-6 polymorphisms in severe and mild cases of COVID-19 in a Chinese population. The variant haplotype C-T-T, represented by rs1800796, rs1524107, and rs2066992 at the IL-6 locus, was reduced in patients with severe illness; in contrast, carriers of the wild-type haplotype G-C-G had higher risk of severe illness. Mechanistically, the protective variant haplotype lost CTCF binding at the IL-6 intron and responded poorly to inflammatory stimuli, which may protect the carriers from hyperinflammation in response to acute SARS-CoV-2 infection. These results point out the possibility that IL-6 genotypes underlie the differential viral virulence during the outbreak of COVID-19. The risk loci we identified may serve as a genetic marker to screen high-risk COVID-19 patients.

Studies on preparation and photodynamic mechanism of chlorin P6-13,15-N-(cyclohexyl)cycloimide (Chlorin-H) and its antitumor effect for photodynamic therapy in vitro and in vivo.

Photodynamic therapy (PDT) represents a promising method for treatment of cancerous tumors. The chemical and physical properties of used photosensitizer play key roles in the treatment efficacy. In this study, a novel photosensitizer, Chlorin-H [-13,15-N-(cyclohexyl)cycloimide] which displayed a characteristic long wavelength absorption peak at 698nm was synthesized. Following flash photolysis with 355nm laser, Chlorin-H is potent to react with O(2) and then produce (1)O(2). This finding indicates that Chlorin-H takes its effects through type II mechanism in PDT. Generally, Chlorin-H is localized in mitochondria and nucleus of cell. After light irradiation with 698nm laser, it can kill many types of cell, inhibit cell proliferation and colony formation, suppress cancer cell invasiveness and trigger apoptosis via the mitochondrial pathway in A549 cells in vitro. In addition, Chlorin-H-PDT can destroy A549 tumor in nude mice and a necrotic scab was formed eventually. The expression levels of many genes which regulated cell growth and apoptosis were determined by RT-PCR following Chlorin-H-PDT. The results showed that it either increased or decrease. Among which, the expression level of TNFSF13, a member of tumor necrosis factor superfamily, increased significantly. Silencing of TNFSF13 caused by RNA interference decreased the susceptibility of A549 cells to Chlorin-H-PDT. In general, Chlorin-H is an effective antitumor photosensitizer in vitro and in vivo and is worthy of further study as a new drug candidate. TNFSF13 will be an important molecular target for the discovery of new photosensitizers.

Chromatin topology reorganization and transcription repression by PML-RARα in acute promyeloid leukemia.

BACKGROUND: Acute promyeloid leukemia (APL) is characterized by the oncogenic fusion protein PML-RARα, a major etiological agent in APL. However, the molecular mechanisms underlying the role of PML-RARα in leukemogenesis remain largely unknown. RESULTS: Using an inducible system, we comprehensively analyze the 3D genome organization in myeloid cells and its reorganization after PML-RARα induction and perform additional analyses in patient-derived APL cells with native PML-RARα. We discover that PML-RARα mediates extensive chromatin interactions genome-wide. Globally, it redefines the chromatin topology of the myeloid genome toward a more condensed configuration in APL cells; locally, it intrudes RNAPII-associated interaction domains, interrupts myeloid-specific transcription factors binding at enhancers and super-enhancers, and leads to transcriptional repression of genes critical for myeloid differentiation and maturation. CONCLUSIONS: Our results not only provide novel topological insights for the roles of PML-RARα in transforming myeloid cells into leukemia cells, but further uncover a topological framework of a molecular mechanism for oncogenic fusion proteins in cancers.

MIA-Sig: multiplex chromatin interaction analysis by signal processing and statistical algorithms.

The single-molecule multiplex chromatin interaction data are generated by emerging 3D genome mapping technologies such as GAM, SPRITE, and ChIA-Drop. These datasets provide insights into high-dimensional chromatin organization, yet introduce new computational challenges. Thus, we developed MIA-Sig, an algorithmic solution based on signal processing and information theory. We demonstrate its ability to de-noise the multiplex data, assess the statistical significance of chromatin complexes, and identify topological domains and frequent inter-domain contacts. On chromatin immunoprecipitation (ChIP)-enriched data, MIA-Sig can clearly distinguish the protein-associated interactions from the non-specific topological domains. Together, MIA-Sig represents a novel algorithmic framework for multiplex chromatin interaction analysis.

A functional variant in the transcriptional regulatory region of gene LOC344967 cosegregates with disease phenotype in familial nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma-associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism -32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The -32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this -32A variant creates an activator protein (AP-1)-binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the -32G/A genotype compared with nasopharyngeal carcinoma cells with the -32G/G genotype. Collectively, these results provide evidence that the -32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied.

Haplotype of gene Nedd4 binding protein 2 associated with sporadic nasopharyngeal carcinoma in the Southern Chinese population.

BACKGROUND: Bcl-3 as an oncoprotein is overexpressed in nasopharyngeal carcinoma (NPC). Nedd4 binding protein 2 (N4BP2), which is located in the NPC susceptibility locus, is a Bcl-3 binding protein. This study is aimed to explore the association between N4BP2 genetic polymorphism and the risk of NPC. METHODS: We performed a hospital-based case-control study, including 531 sporadic NPC and 480 cancer-free control subjects from southern China. PCR-sequencing was carried out on Exons, promoter region and nearby introns of the N4BP2 gene. The expression pattern of N4BP2 and Bcl-3 was also analyzed. RESULTS: We observed a statistically significant difference in haplotype blocks ATTA and GTTG between cases and controls. In addition, three novel SNPs were identified, two of which were in exons (loc123-e3l-snp2, position 39868005, A/G, Met171Val; RS17511668-SNP2, position 39926432, G/A, Glu118Lys), and one was in the intron6 (RS794001-SNP1, position 39944127, T/G). Moreover, N4BP2 was at higher levels in a majority of tumor tissues examined, relative to paired normal tissues. CONCLUSION: These data suggest that haplotype blocks ATTA and GTTG of N4BP2 is correlation with the risk of sporadic nasopharyngeal carcinoma in the Southern Chinese population and N4BP2 has a potential role in the development of NPC.

Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions.

Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.

The expression of p63 is associated with the differential stage in nasopharyngeal carcinoma and EBV infection.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is common among Southern Chinese and the main histology is the undifferentiated carcinoma associated with Epstein-Barr virus (EBV) infection. p63 is a recently proved member of the p53 family based on the structural similarity to p53, but its function in NPC is still unknown. This study was aimed to investigate the association between p63 and NPC. RESULTS: p63 was expressed in 100% (202/202) of nasopharyngeal carcinoma (NPC) tissues but not in 29 nasopharynx inflammation and 17 non-cancerous nasopharyngeal epidermises on a tissue microarray by immunohistostaining. Further investigation suggested that the p63 expression was associated with the differential stage of NPC: p63 strong staining in Keratinizing squamous cell carcinoma, differentiated non-keratinizing NPC and undifferentiated non-keratinizing NPC presented the percentage of 5/8 (62.5%), 43/48 (92.5%) and 50/50 (100%), respectively. A significant difference (p = 0.001) existed between the keratinizing and non-keratinizing groups. No pathogenic mutations were detected in p63 gene in 12 primary NPC tissues and matched peripheral blood lymphocytes (PBL). Half-life measurement study revealed distinct stability of p63 protein in the different cell lines, especially between the carcinoma cell lines with EBV infection and the non-cancerous cell lines. The results of immunoprecipitation suggested a direct interaction between Epstein-Barr virus nuclear antigen 5 (EBNA-5) and p63 protein in NPC, and this binding would increase the stability of p63. CONCLUSION: Our data suggested p63 might be used as an adjunct diagnostic marker of NPC and contributed a new way to understand the contribution of the EBV in the pathogenesis of NPC.